1. Field of the Invention
This invention relates to a method for preferentially extracting and distinguishing infectious norovirus from inactivated norovirus.
2. Description of the Related Art
Human norovirus is the most frequent cause of foodborne illness in the United States (Anon, Morbidity and Mortality Weekly Report, 59:973-979, 2010), and is spread by consumption of contaminated food or water. Noroviruses, previously called Norwalk-like viruses (NLV), are small, round viruses within the calicivirus family and are important viral pathogens that cause acute gastroenteritis, the second most common illness in the United States. Norovirus illness is normally a mild to moderate illness that develops 1-2 days after infection by person-to-person transmission, surface contamination, or by contaminated food or water and the illness lasts for 24 to 60 hours. Symptoms include nausea, vomiting, diarrhea, abdominal pain, and upon occasion headache and low fever. Severe illness, although uncommon, may require hospitalization. Particularly large epidemic outbreaks of illness have occurred following consumption of contaminated water or uncooked foods, such as salads or sliced deli meats and shellfish, such as clams, cockles, and oysters.
Norovirus have been refractory to reliable propagation in vitro (Duizer et al., Journal of General Virology, 85:79-87, 2004) and there are no practical in vivo assays. Consequently, most methods for detection of norovirus contamination in foods utilize molecular methods such as RT-PCR, targeting the viral RNA genome. While primer specificity has improved dramatically in recent years, and RT-PCR methods are now quite sensitive, these techniques have drawbacks. First, successful RT-PCR requires a relatively pure RNA template, free of molecular amplification inhibitors, necessitating the use of sophisticated biochemical extraction techniques. Furthermore, traditional RT-PCR cannot distinguish between RNA derived from an infectious virus or from an inactivated virus (Richards, J. Food Prot, 62:691-697, 1999). While chemically-altered or highly fragmented RNA may not be successfully amplified and ruptured virus particles leave viral RNA vulnerable to rapid environmental degradation, intact virus particles inactivated as a consequence of damage to capsid or other proteins will contain intact RNA despite being unable to initiate an infection in vivo. Essentially, this creates the potential of a false positive test where the detected virus is not a threat to public health.
For norovirus, it is known that the RNA isolated from virions is sufficient to initiate replication of the virus (Guix et al., J. Virol., 81:12238-12248, 2007). Therefore, inactivation mechanisms that target virus proteins must either perturb or rupture the virus capsid, or alter other structural proteins sufficiently to disrupt the early phases of the viral life cycle before release of viral RNA into the cytosol of the infected cell such as for example, attachment, penetration, or uncoating phases of infection.
Norovirus are known to bind to histo-blood group antigens (HBGAs) on the surface of human cells with different norovirus strains recognizing specific variable antigens expressed by subsets of the human population, which potentially explains why some individuals are susceptible to certain strains and resistant to others (Cao et al., J. Virol., 81:5949-5957, 2007; Donaldson et al., Immunol. Rev., 225:190-211, 2008; Tan and Jiang, Trends Microbiol., 13:285-293, 2005). In addition to demonstrating that histo-blood group antigens can be used to concentrate human norovirus, Tian et al. (J. Appl. Microbiol., 109:1753-1762, 2010) demonstrated that recombinant norovirus-like particles readily bind to the surface of swine duodenum (Tian et al., Res. Vet. Sci., 83:410-418, 2007). Subsequently, Tian et al (Appl. Environ. Microbiol., 74:4271-4276, 2008) demonstrated that when porcine gastric mucin was coupled to magnetic beads (PGM-MB), these beads could be used to expediently extract different strains of norovirus from foods, binding 100% of the GI and 85% of the GII norovirus strains tested.
Virus inactivation methods, which are known to substantially target capsid proteins, include thermal methods such as pasterurization, ultraviolet (UV) light inactivation, high pressure processing (HPP) and chlorination. Nuanualsuwan and Cliver (J. Virol. Methods, 104:217-225, 2002) used protein kinase K and RNase A applied after inactivation by chlorine, ultraviolet light, or 72° C. heat treatments to destroy the inactivated virion and the genomic RNA of poliovirus (PV), hepatitis A virus (HAV), and feline calcivirus. This acquired sensitivity to proteinase K infers alteration of the protein capsid structure, resulting in cleavage of capsid proteins, destruction of capsid integrity by proteinase, and subsequent destruction of the virus genome by RNase A. On this basis, it was suggested by the reference that this treatment could potentially be used to avoid positive RT-PCR results from the presence of inactivated viruses. It was also noted that for thermal and UV inactivation, or hypochlorite treatments of FCV, HAV, and PV, loss of infectivity was usually accompanied by the loss of virus attachment to its homologous cellular receptor (Nuanualsuwan and Cliver, 2002, supra; Appl. Environ. Microbiol, 69:350-357, 2003). More recently, Parshionikar et al. (Appl. Environ. Microbiol., 76:4318-4326, 2010) reported that 72° C. treated norovirus is rendered non-detectable by RT-PCR via reaction and modification of encapsulated RNA with propidium monoazide.
Given the relative difficulties of assaying for inactivation of human norovirus, there is a paucity of data confirming inactivation conditions for these virus strains. Conformational stability studies with recombinantly-expressed norovirus-like particles (VLPs), which are in essence empty norovirus capsids, demonstrated alterations of protein structure at temperatures above 60° C. (Ausar et al., J. Biol. Chem., 281:19478-19488, 2006). Using plaque assay, temperature inactivation at 60° C. or higher was observed for the genetically-related research surrogate, murine norovirus (MNV; Baert et al., Appl. Environ. Microbiol., 74:543-546, 2008). High pressure processing, another protein-targeting treatment at 6° C. is capable of inactivating at least 4-log10 of GI.1 Norwalk strain (Leon et al, Appl. Environ. Microbiol. 77:5476-5482, 2011). Also, it has been demonstrated that high pressure-treated MNV becomes deficient for binding to the surface of its host cells (Tang et al., Int. J. Food Microbiol., 137:186-189, 2010). Although the degree to which UV irradiation inactivates norovirus strains has not been determined, UV inactivation studies have shown that a UV dose of 25 mJ/cm2 inactivated 3.6-log10 PFU of MNV (Lee et al., Appl. Environ. Microbiol., 74:2111-2117, 2008).
There remains a need for a method for separating and extracting norovirus virions that are potentially infectious for subsequent RT-PCR analysis. The present invention described below is directed to a method for separating and extracting norovirus virions that are potentially infectious from inactivated strains of norovirus which is different from prior art methods.